Vol. 26, No. 3
INFECTION AND IMMUNITY, Dec. 1979, p. 1014-1019 0019-9567/79/12-1014/06$02.00/0
Mannose-Sensitive Stimulation of Human Leukocyte Chemiluminescence by Escherichia coli DENNIS F. MANGANt AND IRVIN S. SNYDER*
Department of Microbiology, West Virginia University Medical Center, Morgantown, West Virginia 26506 Received for publication 11 September 1979
Escherichia coli organisms with mannose-sensitive adherence factors (adhesins) are known to associate with human peripheral leukocytes (WBCs) in vitro in the absence of serum. To determine whether the WBC respiratory burst is activated during this interaction with E. coli, WBC chemiluminescence was measured. E. coli with mannose-sensitive adhesins stimulated a sharp burst of chemiluminescence which peaked 15 to 30 min after the bacteria and WBCs were mixed. Stimulation of chemiluminescence could be abrogated by including 10 mM a-methyl-D-mannoside in the test suspension. The addition of a-methyl-Dmannoside up to 20 min after the E. coli and WBCs were combined caused a rapid decrease in chemiluminescence. E. coli stimulation of chemiluminescence could not be inhibited by pretreating the WBCs with purified type 1 pili (fimbriae). E. coli lacking mannose-sensitive adhesins failed to stimulate chemiluminescence. The results emphasize the importance of mannose-sensitive adhesins in the association of E. coli with WBCs and suggest that the E. coli-WBC interaction system may be a useful tool for studying the mechanisms involved in the activation of the respiratory burst during phagocytosis.
Interaction of an appropriate stimulus with a phagocytic leukocyte (WBC) triggers a cascade of enzymatic reactions which is referred to as a "respiratory burst" (19). The respiratory burst is characterized by a dramatic increase in 02 consumption by the "activated" phagocytes. An oxidase enzyme located on the cell plasma membrane (12, 17, 18) catalyzes a one-electron reduction of 02 to form 02-, the superoxide ion. 02. is then converted into additional reactive oxidants, including singlet oxygen (102) and the hydroxyl radical (OH.) (3, 20, 26). 02-, 102, and OH. are high-energy compounds which release their energy in the form of light during the oxidation of a suitable substrate or during return to ground-state 02 (1, 2, 9). The light emitted is referred to as chemiluminescence (CL) and can be measured in a modified liquid scintillation spectrometer (1, 2). Recent studies by us (22) and by other investigators (4, 25) showed that Escherichia coli organisms associated with and are phagocytized by human WBCs in vitro in the absence of serum opsonins. The interaction between E. coli and the WBCs appears to be mediated by type 1 pili (fimbriae) (7, 8), a mannose-sensitive (MS) adherence factor (adhesin). The basis for this is that (i) only piliated E. coli are phagocytized t Present address: Department of Oral Biology, Dental Research Institute, University of Michigan, Ann Arbor, MI 48109.
and (ii) the association is inhibited by low concentrations of D-mannose or D-mannose derivatives (e.g., a-methyl-D-mannopyranoside [aMM]) but not by several other saccharides. However, we have repeatedly failed to block E. coli-WBC interaction by pretreating the WBCs with purified type 1 pili (22), suggesting that the E. coli may possess another MS adhesin which mediates the association with the WBCs. Eshdat et al. (16) have reported the isolation from E. coli of a "mannose specific lectin" which is functionally identical to type 1 pili, but which differs from type 1 pili in amino acid composition and subunit molecular weight. Whether this lectin material is involved in E. coli-WBC interaction has not been determined. To further study the association between E. coli and human WBCs, we used CL as a measurement of the bacterium-phagocyte interaction. The results show that E. coli with MS adhesins stimulate CL and that the stimulation can be inhibited by aMM. E. coli stimulation of CL cannot, however, be inhibited by pretreating the WBCs with purified pili. MATERIALS AND METHODS E. coli All strains used in this study, with the exception of strain Bam, were clinical isolates from the West Virginia University Hospital. E. coli Bam, the prototype strain for type 1 pili, was kindly donated by Charles C. Brinton, Jr., University of Pittsburgh,
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VOL. 26, 1979
MS STIMULATION OF WBC CL BY E. COLI
Pittsburgh, Pa. E. coli strain 19 was isolated from a urinary tract infection. Isolates were identified as E. coli by Gram stain and by biochemical characteristics (15). Preparation of E. coli cultures for WBC studies. Strains of E. coli were grown under conditions which stimulate or depress type 1 pili production (P) and MS hemagglutination (HA) of guinea pig erythrocytes (RBCs) (13). The methods used were described in a recent report (22). In brief, P+, HA+ cultures (e.g., 19+, Bam+) were derived by repeated subculture of the E. coli strain in Trypticase soy broth with dextrose (TSB; BBL Microbiology Systems, Cockeysville, Md.) at 370C without agitation. P-, HA- cultures (e.g., 19-, Bam-) were derived by repeated subculture of the E. coli strain in TSB at 300C with agitation. The bacterial cells were washed and suspended in 0.01 M phosphate-buffered 0.15 M NaCl, pH 7.4 (PBS), to the desired concentration. Heat and formaldehyde pretreatment of E. coli Before being incubated with WRBCs, E. coli cells were pretreated with heat (56, 75, or 1000C) or 1% formaldehyde as previously described (22). HA assays. HA was determined by slide agglutination with 2.5% guinea pig RBCs in 0.4% bovine serum albumin (BSA)-PBS. E. coli cultures which were HA+ in the presence of 50 mM dextrose or sucrose (Mallinckrodt, St. Louis, Mo.), but HA- in the presence of 50 mM aMM (Sigma Chemical Co., St. Louis, Mo.), were labeled MS. HA titers were determined in plastic round-bottom microtiter plates. Serial twofold dilutions of a suspension of E. coli (optical density at 625 nm = 0.70) were made in 50 tl of PBS. To each well, 50 pI of a 0.25% suspension of guinea pig RBCs in 1% BSA-PBS was added. The plates were sealed with acetate tape to prevent drying, mixed by tapping for 30 s, and incubated at SOC for 18 h. Titers were defined as the reciprocal of the last dilution giving a complete HA reaction. Purification of type 1 pili. Pili were purified from E. coli 19+ and Bam+ as previously described (22). Purified pili appeared free from contaminating substances under electron microscopy and retained the ability to agglutinate guinea pig RBCs (22). WBC preparation. For a given experiment, all tests were performed with WBCs obtained from a single human blood donor. All glassware, except pipettes, was siliconized (Dri-Film SC87; Pierce Chemical Co., Rockford, Ill.). Peripheral venous blood was collected in heparin (143 U/10-ml tube). WBCs were separated from RBCs by dextran sedimentation (6) and washed in 0.1% BSA-PBS. Residual RBCs were removed by hypotonic saline lysis (10). After an additional wash with 0.1% BSA-PBS, the WBCs were resuspended and adjusted to the desired concentration in 0.4% BSAPBS. Suspensions typically contained 65 to 80% polymorphonuclear neutrophils. CL. CL was measured in a Packard scintillation spectrometer, model 3320, using the out-of-coincidence summation mode, a window setting of 0 to infinity, and 100% gain (1, 2). All procedures were performed under dim light to minimize background light emission. Sterile, siliconized, glass scintillation
1015
vials were dark adapted for at least 20 h before use. Each vial, containing 3.5 ml of PBS and 1.0 ml of the test suspension or PBS (negative-stimulus control), was warmed to 370C in a rotating water bath. After 5 min, background CL measurements were made. One milliliter of a WBC suspension (5.0 x 10' WBCs/ml) was added to each vial, staggering the addition at 35-s intervals so that CL measurements for all vials were at equal intervals after the addition of the WBCs (zero time). Vials were held in a 370C water bath between CL measurements. Approximately 10 s before each CL measurement, the vial was removed from the water bath and dried. CL was then measured for 20 s, and the vial was then quickly returned to the water bath. Opsonized zymosan. Ten milligrams of zymosan (Zymosan A, Sigma) was added to 1.0 ml of fresh human serum, and the suspension was incubated at 370C for 30 min. The opsonized zymosan particles were centrifuged at 2,000 x gfor 10 min at 50C, washed once in PBS, and finally suspended in PBS to a concentration of 5 mg/ml.
RESULTS E. coli stimulation of CL. Since prior studies showed that E. coli cells with MS adhesins associated with WBCs in the absence of serum opsonins (4, 22, 25), we tested the ability of these E. coli cells to stimulate CL. As shown in Fig. 1, WBCs incubated with E. coli possessing MS adhesins (19+, Bam+, 42+, 51+, and 44+) yielded a burst of CL which peaked between 15 and 30 min after the bacteria and WBCs were mixed together. WBCs incubated with E. coli lacking MS adhesins (19- and 51-) or with PBS 0
°
70
2
60 e
8'20 60
/ k
i 9s ~S+ Born~~~~~~~~~~~~~~~1
z~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~2+ 40ADE
MIN AFTER ADDITION OF WBC
FIG. 1. CL from WBCs incubated with several E. coli strains. The bacteria-to- WBC ratio was 300:1. A nonstimulated (PBS) control suspension lacked E. coli. At 60 min, 0.5 ml of a suspension of E. coli 19+ (1.8 x lOP colony-forming units per ml) was added to several vials, and the CL was assayed for an additional 20 min. Each curve represents the CL values from a single test vial during one of five representative experiments.
1016 MANGAN AND SNYDER (no bacteria) did not stimulate CL. In other experiments (data not shown), E. coli 30+ and 22+ (with MS adhesins) stimulated CL, whereas E. coli 22- and 38- (without MS adhesins) failed to stimulate CL. To determine whether the WBCs previously incubated with E. coli or the PBS control could be stimulated further, we added 19+ cells to several test vials (Fig. 1). The addition of 19+ to suspensions already containing 19+ cells did not result in an additional burst of CL. The addition of 19+ to suspensions containing 19- or to the PBS control vial caused large CL bursts. Pretreatment of E. coli 19+ with formaldehyde and heat. Figure 2 shows the effect of various pretreatments on the ability of 19+ to stimulate CL. Pretreatment of 19+ with 1% formaldehyde or heating at 560C for 60 min had no effect on the CL response as compared with untreated 19+ suspensions. Heating organisms at 750C for 60 min or at 100'C for 15 min did not cause CL. Viable E. coli organisms were found in the 560C-treated suspensions, but not in the other treated test suspensions, proving that viable E. coli organisms are not required to stimulate CL. Effect of culture conditions on CL stimulation. From previous experiments, we know that E. coli 19 grown repeatedly in TSB at 370C yielded cultures with high HA titers and stimulated large amounts of CL, whereas E. coli 19 grown repeatedly in TSB at 30'C yielded cultures with low HA titers and failed to stimulate CL. We questioned whether E. coli 19 could be grown so that it had an HA titer between the HA titers of 19+ and 19- cultures, and if so, whether this "intermediate" culture would stimulate an intermediate amount of CL. To test this
INFECT. IMMUN.
possibility, we cultured E. coli 19+ and 19- as described above. The intermediate culture was obtained by a single subculture of 19- in TSB at 370C. Figure 3 shows the CL responses to the three cultures of E. coli 19. The 19+ culture produced the highest peak CL (62,000 counts per 20 s) and had an HA titer of 32 to 64. E. coli 19- cultures did not have detectable HA activity and did not stimulate CL. The intermediate culture has an HA titer of 16 and stimulated a CL burst which was approximately two-thirds the values obtained from 19+. Thus, there appears to be a general positive correlation between HA titer and CL stimulation. CL as a function of E. coli 19+ concentration. Dilutions of E. coli 19+ were added to a constant number of WBCs, and the resulting CL was measured. Increasing the ratio of 19+ to WBCs caused elevated CL bursts which peaked more quickly than lower ratios of bacteria to WBCs (Fig. 4). In this assay, the minimum detectable CL was obtained with a ratio of three 19+ cells per WBC. Inhibition of CL with aMM. aMM inhibits the association of E. coli with WBCs (4, 22). To determine whether E. coli association with the WBCs was necessary to stimulate CL, we added aMM to the CL assay suspensions. Formaldehyde-killed bacteria (see below) were used to eliminate catabolism of the carbohydrate during the 60-min incubation at 370C. t0
60
0
20
40
60
80
MINUTES AFTER ADDITION OF WBC
5
0
10
20
30
75C
40
50
60
MIN AFTER ADDITION OF WBC
FIG. 2. Effect ofpretreatment of E. coli 19+ on CL. E. coli organisms were pretreated with formaldehyde and heated as described in the text. The bacteria-toWBC ratio was 300:1. Each curve represents mean CL values from duplicate test vials during one of two similar experiments.
FIG. 3. CL from WBCs incubated with different cultures of E. coli 19. The bacteria-to- WBC ratio was 300:1. The PBS control lacked bacteria. Each curve represents mean CL values from duplicate test vials during a single experiment. E. coli 19 organisms were serially subcultured as follows: four times in TSB at 37°C (0); three times in TSB at 370C, followed by a single subculture in TSB at 370C (LI); or four times in TSB at 370C (0). Control vials received PBS alone (). Each curve represents mean CL values from duplicate test vials during one of two similar experiments.
MS STIMULATION OF WBC CL BY E. COLI 1017
VOL. 26, 1979
7O60 150 10 50
O
10
.
20
30
.
40
50
6
30.1 w W~~~~~ 2
ulate CL even when tested in concentrations as high as 200 Ag/ml. Pretreatment of WBCs with purified pill. An attempts was made to block 19+-induced CL by pretreating the WBCs (5 x 106/ml) with purified 19+ pili (187.5 [Lg/ml) for 15 min at room temperature (R0). When stimulated by 19+, pili-pretreated WBCs yielded a slightly higher peak CL (40,250 counts per 20 s) as compared with PBS-pretreated WBCs (38,000 counts per 20 s). Similar results were obtained 35
W
z 225
~~~~~~~~300-1 with 10mnM aMM
10
25X m DEXTROSE
a20
CONTROL Z
0
10
40 50 30 MIN AFTER ADDITION OF WBC
20
60
FIG. 4. CL from WBCs incubated with increasing concentrations of E. coli 19+. Bacteria-to- WBC ratios are shown for each curve. The unstimulated (PBS) control lacked E. coli. Each curve represents CL values from a single test vial during one of three representative experiments.
As little as 10 mM aMM completely inhibited CL stimulation by 19+ (Fig. 4). Neither dextrose nor sucrose (50 mM) blocked CL, whereas an equal concentration of aMM prevented CL (Fig. 5). In fact, 25 mM dextrose stimulated the CL burst elicited by 19+. The effect of aMM on CL stimulated by opsonized zymosan particles was also tested. Zymosan (5 mg/ml) caused peak CL of 211,500 counts per 20 s in the presence of 63.6 mM aMM and 224,500 counts per 20 s in the absence of aMM. WBCs incubated with aMM alone gave no CL. Thus, aMM does not appear to nonspecifically interfere with or stimulate WBC CL. Addition of aMM after E. coli association with WBCs. To determine whether CL can be stopped once the bacteria have associated with the WBCs, we added aMM to the test vials at various time intervals during the incubation. As shown in Fig. 6, the addition of 50 mM aMM within 5 min after the bacteria and WBCs were mixed completely prevented CL. After 5 min, the addition of aMM inhibited further increases in CL and caused a rapid decline in residual light emission. Purified pili. Several CL assays were done to determine whether purified 19+ and Bam+ pili could stimulate CL. However, pili failed to stim-
mM DEXTROSE
'j 5
5O/m
mm a MM 25mM aRM
50
10
D
o
SUCROSE
25 nM SUCROSE
UT
5. 5-
10
20
50 40 30 MIN AFTER ADDITION OF WBC
60
FIG. 5. Effect of saccharides on CL. E. coli Bam+ organisms were killed with 1% formaldehyde before the assay. The bacteria-to-WBC ratio was 180:1. Each curve represents the CL values from a single test vial during one of three similar experiments. 0
0
-t
I?z z -i
IL 0
10
20
50 60 40 30 MIN AFTER ADDITION OF WBC
70
FIG. 6. Addition of aMM at intervals during the incubation of WBCs with E. coli Bam+. E. coli organisms were killed with 1% formaldehyde before the assay. The bacteria-to-WBC ratio was 180:1. At the times indicated above each curve, aMM in PBS (0.27 ml) was added to the vials to a final concentration of 50 mM. The control suspension contained an equal volume of PBS added at zero time. Each curve represents the CL values from a single test vial during one of two representative experiments.
1018 MANGAN AND SNYDER with WBCs pretreated with purified Bam+ pili (133.3 ,ug/ml) and stimulated with Bam+. Pilipretreated and control WBCs failed to emit CL when incubated with 19- or Bam-. Unstimulated WBCs pretreated with pili did not emit CL (see above). DISCUSSION In previous studies, piliated E. coli with MS adhesins associated with and were killed by human WBCs in the presence of serum opsonins (22, 25). We further showed that piliated, but not nonpiliated, E. coli organisms caused a release of lysosomal enzymes from the WBCs without damaging the WBCs (22). In this communication, we extend our observations on the E. coli-WBC interaction by reporting that the association of these bacteria with the WBCs activates oxidative metabolism in the WBCs as detected by enhanced CL. E. coli organisms which lack MS adhesins failed to stimulate CL. WBCs incubated with E. coli 19- and challenged with strain 19+ yielded a CL burst of intensity equal to that obtained from WBCs incubated with PBS and stimulated with 19+. This indicates that the absence of CL from WBCs incubated with E. coli organisms without MS adhesins resulted from a lack of stimulation rather than from an inhibition or quenching of CL. WBCs incubated with 42+ yielded CL and could be further stimulated by 19+ to yield additional CL. This suggests that 19+ cultures either have more CL-stimulating cells or have cells with quantitatively more CL-stimulating activity than do 42+ cultures. This result also indicates that WBCs can detect a quantitative difference in CL-stimulating activity in E. coli cultures and respond to further stimulation with a second burst of CL. Concanvalin A (ConA), a plant lectin, binds to MS receptors on WBCs (21) and stimulates oxygen metabolism (24) and CL (unpublished data). Thus, one might speculate that the stimulation of WBC respiratory activity by ConA and E. coli MS adherence factors is mediated through common receptors by similar mechanisms.
Many of the biological effects associated with ConA appear to result from the ability of this multivalent lectin to aggregate mobile glycoprotein receptors into micropatches on the surface of the cells (11, 14). Clustering of ConA receptors on phagocytes has been noted (5), and Romeo et al. (24) speculated that the formation of receptor patches activates the oxidase enzyme involved in initiating the respiratory burst. In a similar fashion, E. coli organisms with MS adhesins may bind to several receptors simultane-
INFECT. IMMUN.
ously in one small area of the WBC surface and form a receptor patch. Isolated type 1 pili were unable to stimulate CL. The orientation of the pili during adherence to the WBC surface is unknown, but may be in such a manner that they fail to form the necessary clustering of receptors needed to trigger the oxidase enzyme. Alternatively, E. coli organisms may possess a second MS adhesin which mediates the association with WBCs and activates CL. Our prior finding that type 1 pili failed to inhibit the attachment of E. coli to WBCs (22), coupled with the date in this paper which show that CL stimulation by piliated E. coli is not inhibited to pili, tends to support the conclusion of a second adhesin. In this regard, Silverblatt et al. (25) obtained a poor correlation between the number of pili on several E. coli strains and the susceptibility of these bacteria to phagocytosis. Furthermore, Eshdat et al. (16) have recently isolated a mannose-specific lectin from E. coli which is functionally identical to type 1 pili, but which has an amino acid composition and subunit molecular weight which are different from type 1 pili. Whether our strains of E. coli possess this bacterial lectin and the role that the lectin has in the association of E. coli with WBCs remain to be determined. D-Mannose and aMM reversibly inhibit MS adherence of E. coli to eucaryotic cells (23). We found that aMM added to Bam+-WBC suspensions incubated at 370C for up to 20 min immediately interrupted CL, and subsequent light emission rapidly returned to control levels. Romeo and associates (24) reported that the oxidative stimulation of phagocytes by ConA could likewise to immediately reversed by the addition of a-methyl-D-glucopyranoside, a saccharide which reversibly inhibits ConA adherence. Therefore, these reports, suggest that the continual attachment of the stimulus to the WBC surface is required for the activation of the respiratory burst. In previous studies (22), we found that after incubation at 370C for 15 min, many E. coli organisms which associate with WBCs are phagocytized (ingested) and are not susceptible to displacement by the aMM treatment. The data from the CL and ingestion studies, therefore, suggest that only E. coli attached to and pertubating the outer membrane of the WBC participates in the stimulation of CL. This conclusion supports the evidence which indicates that CL results from the stimulation of a surface enzyme (12, 17, 18). Furthermore, the data suggest that the E. coli organisms internalized before the addition of aMM are incapable of triggering the oxidase enzyme responsible for initiating the respiratory burst. An explanation for this is presently unavailable, but it may be
MS STIMULATION OF WBC CL BY E. COLI 1019
VOL. 26, 1979
that the oxidase enzyme either may not be internalized as the cell membrane invaginates during phagocytosis or may be inactive when internalized. Further studies are necessary to determine the mechanism by which the oxidase enzyme is activated during the adherence of E. coli to MS receptors on the WBC membrane. Additional characterization of the MS adhesins on E. coli will undoubtedly lead to a better understanding of the E. coli-WBC interaction. More importantly, this particular bacteriumphagocyte relationship deserves special attention because (i) it occurs in the absence of serum opsonins, (ii) it involves a specific interaction between a bacterial adherence factor and a eucaryotic cell receptor, and (iii) it is readily reversible by a low concentration of aMM. The E. coli-WBC interaction system may, therefore, be a very useful tool for studying the cellular events involved in phagocytosis and the stimulation of CL. ACKNOWLEDGMENTS This work was supported by Biomedical Research Support Grant SO 7 RR 05433 17 and by the West Virginia University Medical Corp. The secretarial assistance of Elizabeth Rocovich is gratefully acknowledged.
LITERATURE CITED 1. Allen, R. C., R. L. Stjernholm, and R. H. Steele. 1972. Evidence for the generation of an electronic excitation state(s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem. Biophys. Res. Commun. 47:679-684. 2. Allen, R. C., S. J. Yevich, R. W. Orth, and R. H. Steele. 1974. The superoxide anion and singlet molecular oxygen: their role in the microbicidal activity of the polymorphonuclear leukocyte. Biochem. Biophys. Res. Commun. 60:909-917. 3. Babior, B. M., R. S. Kipnes, and J. T. Curnutte. 1973. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J. Clin. Invest. 52:741-744. 4. Bar-Shavit, Z., I. Ofek, R. Goldman, D. Mirelman, and N. Sharon. 1977. Mannose residues on phagocytes as receptors for the attachment of Escherichia coli and Salmonella typhi. Biochem. Biophys. Res. Commun. 78:455-460. 5. Berlin, R. 1972. Effect of Concanavalin A on phagocytosis. Nature (London) New Biol. 235:44-45. 6. Boyum, A. 1968. Isolation of leukocytes from human blood: further observations. Methylcellulose, dextran, and ficoll as erythrocyte-aggregating agents. Scand. J. Clin. Lab. Invest. 21(Suppl. 9):31-50. 7. Brinton, C. C., Jr. 1965. The structure, function, synthesis, and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram negative bacteria. Trans. N.Y. Acad. Sci. 27:1003-1054. 8. Brinton, C. C., Jr. 1967. Contributions of pili to the specificity of the bacterial surface, and a unitary hy-
pothesis of conjugal infectious heredity, p. 37-70. In B. D. Davis and L. Warren (ed.), The specificity of cell surfaces. Prentice-Hall, Inc., Englewood Cliffs, N.J. 9. Cheson, B. D., R. J. Christensen, R. Sperling, B. E. Kohler, and B. M. Babior. 1976. The origin of the chemiluminescence of phagocytosing granulocytes. J. Clin. Invest. 58:789-796. 10. Clark, R. A., and H. R. Kimball. 1971. Defective granulocyte chemotaxis in the Chediak-Higashi syndrome. J. Clin. Invest. 50:2645-2652. 11. Cunningham, B. A., B. A. Sela, I. Yahara, and G. M. Edelman. 1976. Structure and activities of lymphocyte mitogens, p. 13-30. In J. J. Openheim and D. L. Rosenstreich (ed.), Mitogens in immunology. Academic Press Inc., New York. 12. Dewald, B., M. Gaggiolini, J. T. Curnutte, and B. M. Babior. 1979. Subcellular localization of the superoxide-forming enzyme in human neutrophils. J. Clin. Invest. 63:21-29. 13. Duguid, J. P., and R. R. Gillies. 1957. Fimbriae and adhesive properties in dysentery bacilli. J. Pathol. Bacteriol. 74:397-411. 14. Edelman, G. M., B. A. Cunningham, G. N. Reeke, Jr., J. W. Becker, M. J. Waxdal, and J. L. Want. 1972. The covalent and three-dimensional structure of Concanavalin A. Proc. Natl. Acad. Sci. U.S.A. 69:25802584. 15. Edwards, P. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed., p. 21-47. Burgess Publishing Co., Minneapolis, Minn. 16. Eshdat, Y., I. Ofek, Y. Yashour-Gan, N. Sharon, and D. Mirelman. 1978. Isolation of a mannose-specific lectin from Escherichia coli and its role in the adherence of the bacteria to epithelial cells. Biochem. Biophys. Res. Commun. 85:1551-1559. 17. Goldstein, I. M., M. Cerqueria, S. Lind, and H. B. Kaplan. 1977. Evidence that the superoxide-generating system of human leukocytes is associated with the cell surface. J. Clin. Invest. 59:249-254. 18. Goldstein, L. M., D. Roos, H. B. Kaplan, and G. Weissmann. 1975. Complement and immunoglobulins stimulate superoxide production by human leukocytes independently of phagocytosis. J. Clin. Invest. 56:11551163. 19. Karnovsky, M. L. 1962. Metabolic basis of phagocytic activity. Physiol. Rev. 42:143-168. 20. Klebanoff, S. J. 1975. Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Semin. Hematol. 12:117-142. 21. iUs, H., and N. Sharon. 1973. The biochemistry of plant lectins (phytohemagglutinins). Annu. Rev. Biochem. 42:541-574. 22. Mangan, D. F., and L. S. Snyder. 1979. Mannose-sensitive interaction of Escherichia coli with human peripheral leukocytes in vitro. Infect. Immun. 26:520-527. 23. Ofek, I., E. H. Beachey, and N. Sharon. 1978. Surface sugars of animal cells as determinants of recognition in bacterial adherence. Trends Biochem. Sci. 3:159-160. 24. Romeo, D., G. Zabucchi, and F. Rossi. 1973. Reversible metabolic stimulation of polymorphonuclear leukocytes and macrophages by Concanavalin A. Nature (London) New Biol. 243:111-112. 25. Silverblatt, F. J., J. S. Dreyer, and S. Schauer. 1979. Effect of pili on susceptibility of Escherichia coli to phagocytosis. Infect. Immun. 24:218-223. 26. Stossel, T. P. 1974. Phagocytosis. N. Engl. J. Med. 290: 717-724, 774-780, 833-838.
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Mannose-Sensitive Stimulation of Human Leukocyte Chemiluminescence by Escherichia coli DENNIS F. MANGANt AND IRVIN S. SNYDER*
Department of Microbiology, West Virginia University Medical Center, Morgantown, West Virginia 26506 Received for publication 11 September 1979
Escherichia coli organisms with mannose-sensitive adherence factors (adhesins) are known to associate with human peripheral leukocytes (WBCs) in vitro in the absence of serum. To determine whether the WBC respiratory burst is activated during this interaction with E. coli, WBC chemiluminescence was measured. E. coli with mannose-sensitive adhesins stimulated a sharp burst of chemiluminescence which peaked 15 to 30 min after the bacteria and WBCs were mixed. Stimulation of chemiluminescence could be abrogated by including 10 mM a-methyl-D-mannoside in the test suspension. The addition of a-methyl-Dmannoside up to 20 min after the E. coli and WBCs were combined caused a rapid decrease in chemiluminescence. E. coli stimulation of chemiluminescence could not be inhibited by pretreating the WBCs with purified type 1 pili (fimbriae). E. coli lacking mannose-sensitive adhesins failed to stimulate chemiluminescence. The results emphasize the importance of mannose-sensitive adhesins in the association of E. coli with WBCs and suggest that the E. coli-WBC interaction system may be a useful tool for studying the mechanisms involved in the activation of the respiratory burst during phagocytosis.
Interaction of an appropriate stimulus with a phagocytic leukocyte (WBC) triggers a cascade of enzymatic reactions which is referred to as a "respiratory burst" (19). The respiratory burst is characterized by a dramatic increase in 02 consumption by the "activated" phagocytes. An oxidase enzyme located on the cell plasma membrane (12, 17, 18) catalyzes a one-electron reduction of 02 to form 02-, the superoxide ion. 02. is then converted into additional reactive oxidants, including singlet oxygen (102) and the hydroxyl radical (OH.) (3, 20, 26). 02-, 102, and OH. are high-energy compounds which release their energy in the form of light during the oxidation of a suitable substrate or during return to ground-state 02 (1, 2, 9). The light emitted is referred to as chemiluminescence (CL) and can be measured in a modified liquid scintillation spectrometer (1, 2). Recent studies by us (22) and by other investigators (4, 25) showed that Escherichia coli organisms associated with and are phagocytized by human WBCs in vitro in the absence of serum opsonins. The interaction between E. coli and the WBCs appears to be mediated by type 1 pili (fimbriae) (7, 8), a mannose-sensitive (MS) adherence factor (adhesin). The basis for this is that (i) only piliated E. coli are phagocytized t Present address: Department of Oral Biology, Dental Research Institute, University of Michigan, Ann Arbor, MI 48109.
and (ii) the association is inhibited by low concentrations of D-mannose or D-mannose derivatives (e.g., a-methyl-D-mannopyranoside [aMM]) but not by several other saccharides. However, we have repeatedly failed to block E. coli-WBC interaction by pretreating the WBCs with purified type 1 pili (22), suggesting that the E. coli may possess another MS adhesin which mediates the association with the WBCs. Eshdat et al. (16) have reported the isolation from E. coli of a "mannose specific lectin" which is functionally identical to type 1 pili, but which differs from type 1 pili in amino acid composition and subunit molecular weight. Whether this lectin material is involved in E. coli-WBC interaction has not been determined. To further study the association between E. coli and human WBCs, we used CL as a measurement of the bacterium-phagocyte interaction. The results show that E. coli with MS adhesins stimulate CL and that the stimulation can be inhibited by aMM. E. coli stimulation of CL cannot, however, be inhibited by pretreating the WBCs with purified pili. MATERIALS AND METHODS E. coli All strains used in this study, with the exception of strain Bam, were clinical isolates from the West Virginia University Hospital. E. coli Bam, the prototype strain for type 1 pili, was kindly donated by Charles C. Brinton, Jr., University of Pittsburgh,
1014
VOL. 26, 1979
MS STIMULATION OF WBC CL BY E. COLI
Pittsburgh, Pa. E. coli strain 19 was isolated from a urinary tract infection. Isolates were identified as E. coli by Gram stain and by biochemical characteristics (15). Preparation of E. coli cultures for WBC studies. Strains of E. coli were grown under conditions which stimulate or depress type 1 pili production (P) and MS hemagglutination (HA) of guinea pig erythrocytes (RBCs) (13). The methods used were described in a recent report (22). In brief, P+, HA+ cultures (e.g., 19+, Bam+) were derived by repeated subculture of the E. coli strain in Trypticase soy broth with dextrose (TSB; BBL Microbiology Systems, Cockeysville, Md.) at 370C without agitation. P-, HA- cultures (e.g., 19-, Bam-) were derived by repeated subculture of the E. coli strain in TSB at 300C with agitation. The bacterial cells were washed and suspended in 0.01 M phosphate-buffered 0.15 M NaCl, pH 7.4 (PBS), to the desired concentration. Heat and formaldehyde pretreatment of E. coli Before being incubated with WRBCs, E. coli cells were pretreated with heat (56, 75, or 1000C) or 1% formaldehyde as previously described (22). HA assays. HA was determined by slide agglutination with 2.5% guinea pig RBCs in 0.4% bovine serum albumin (BSA)-PBS. E. coli cultures which were HA+ in the presence of 50 mM dextrose or sucrose (Mallinckrodt, St. Louis, Mo.), but HA- in the presence of 50 mM aMM (Sigma Chemical Co., St. Louis, Mo.), were labeled MS. HA titers were determined in plastic round-bottom microtiter plates. Serial twofold dilutions of a suspension of E. coli (optical density at 625 nm = 0.70) were made in 50 tl of PBS. To each well, 50 pI of a 0.25% suspension of guinea pig RBCs in 1% BSA-PBS was added. The plates were sealed with acetate tape to prevent drying, mixed by tapping for 30 s, and incubated at SOC for 18 h. Titers were defined as the reciprocal of the last dilution giving a complete HA reaction. Purification of type 1 pili. Pili were purified from E. coli 19+ and Bam+ as previously described (22). Purified pili appeared free from contaminating substances under electron microscopy and retained the ability to agglutinate guinea pig RBCs (22). WBC preparation. For a given experiment, all tests were performed with WBCs obtained from a single human blood donor. All glassware, except pipettes, was siliconized (Dri-Film SC87; Pierce Chemical Co., Rockford, Ill.). Peripheral venous blood was collected in heparin (143 U/10-ml tube). WBCs were separated from RBCs by dextran sedimentation (6) and washed in 0.1% BSA-PBS. Residual RBCs were removed by hypotonic saline lysis (10). After an additional wash with 0.1% BSA-PBS, the WBCs were resuspended and adjusted to the desired concentration in 0.4% BSAPBS. Suspensions typically contained 65 to 80% polymorphonuclear neutrophils. CL. CL was measured in a Packard scintillation spectrometer, model 3320, using the out-of-coincidence summation mode, a window setting of 0 to infinity, and 100% gain (1, 2). All procedures were performed under dim light to minimize background light emission. Sterile, siliconized, glass scintillation
1015
vials were dark adapted for at least 20 h before use. Each vial, containing 3.5 ml of PBS and 1.0 ml of the test suspension or PBS (negative-stimulus control), was warmed to 370C in a rotating water bath. After 5 min, background CL measurements were made. One milliliter of a WBC suspension (5.0 x 10' WBCs/ml) was added to each vial, staggering the addition at 35-s intervals so that CL measurements for all vials were at equal intervals after the addition of the WBCs (zero time). Vials were held in a 370C water bath between CL measurements. Approximately 10 s before each CL measurement, the vial was removed from the water bath and dried. CL was then measured for 20 s, and the vial was then quickly returned to the water bath. Opsonized zymosan. Ten milligrams of zymosan (Zymosan A, Sigma) was added to 1.0 ml of fresh human serum, and the suspension was incubated at 370C for 30 min. The opsonized zymosan particles were centrifuged at 2,000 x gfor 10 min at 50C, washed once in PBS, and finally suspended in PBS to a concentration of 5 mg/ml.
RESULTS E. coli stimulation of CL. Since prior studies showed that E. coli cells with MS adhesins associated with WBCs in the absence of serum opsonins (4, 22, 25), we tested the ability of these E. coli cells to stimulate CL. As shown in Fig. 1, WBCs incubated with E. coli possessing MS adhesins (19+, Bam+, 42+, 51+, and 44+) yielded a burst of CL which peaked between 15 and 30 min after the bacteria and WBCs were mixed together. WBCs incubated with E. coli lacking MS adhesins (19- and 51-) or with PBS 0
°
70
2
60 e
8'20 60
/ k
i 9s ~S+ Born~~~~~~~~~~~~~~~1
z~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~2+ 40ADE
MIN AFTER ADDITION OF WBC
FIG. 1. CL from WBCs incubated with several E. coli strains. The bacteria-to- WBC ratio was 300:1. A nonstimulated (PBS) control suspension lacked E. coli. At 60 min, 0.5 ml of a suspension of E. coli 19+ (1.8 x lOP colony-forming units per ml) was added to several vials, and the CL was assayed for an additional 20 min. Each curve represents the CL values from a single test vial during one of five representative experiments.
1016 MANGAN AND SNYDER (no bacteria) did not stimulate CL. In other experiments (data not shown), E. coli 30+ and 22+ (with MS adhesins) stimulated CL, whereas E. coli 22- and 38- (without MS adhesins) failed to stimulate CL. To determine whether the WBCs previously incubated with E. coli or the PBS control could be stimulated further, we added 19+ cells to several test vials (Fig. 1). The addition of 19+ to suspensions already containing 19+ cells did not result in an additional burst of CL. The addition of 19+ to suspensions containing 19- or to the PBS control vial caused large CL bursts. Pretreatment of E. coli 19+ with formaldehyde and heat. Figure 2 shows the effect of various pretreatments on the ability of 19+ to stimulate CL. Pretreatment of 19+ with 1% formaldehyde or heating at 560C for 60 min had no effect on the CL response as compared with untreated 19+ suspensions. Heating organisms at 750C for 60 min or at 100'C for 15 min did not cause CL. Viable E. coli organisms were found in the 560C-treated suspensions, but not in the other treated test suspensions, proving that viable E. coli organisms are not required to stimulate CL. Effect of culture conditions on CL stimulation. From previous experiments, we know that E. coli 19 grown repeatedly in TSB at 370C yielded cultures with high HA titers and stimulated large amounts of CL, whereas E. coli 19 grown repeatedly in TSB at 30'C yielded cultures with low HA titers and failed to stimulate CL. We questioned whether E. coli 19 could be grown so that it had an HA titer between the HA titers of 19+ and 19- cultures, and if so, whether this "intermediate" culture would stimulate an intermediate amount of CL. To test this
INFECT. IMMUN.
possibility, we cultured E. coli 19+ and 19- as described above. The intermediate culture was obtained by a single subculture of 19- in TSB at 370C. Figure 3 shows the CL responses to the three cultures of E. coli 19. The 19+ culture produced the highest peak CL (62,000 counts per 20 s) and had an HA titer of 32 to 64. E. coli 19- cultures did not have detectable HA activity and did not stimulate CL. The intermediate culture has an HA titer of 16 and stimulated a CL burst which was approximately two-thirds the values obtained from 19+. Thus, there appears to be a general positive correlation between HA titer and CL stimulation. CL as a function of E. coli 19+ concentration. Dilutions of E. coli 19+ were added to a constant number of WBCs, and the resulting CL was measured. Increasing the ratio of 19+ to WBCs caused elevated CL bursts which peaked more quickly than lower ratios of bacteria to WBCs (Fig. 4). In this assay, the minimum detectable CL was obtained with a ratio of three 19+ cells per WBC. Inhibition of CL with aMM. aMM inhibits the association of E. coli with WBCs (4, 22). To determine whether E. coli association with the WBCs was necessary to stimulate CL, we added aMM to the CL assay suspensions. Formaldehyde-killed bacteria (see below) were used to eliminate catabolism of the carbohydrate during the 60-min incubation at 370C. t0
60
0
20
40
60
80
MINUTES AFTER ADDITION OF WBC
5
0
10
20
30
75C
40
50
60
MIN AFTER ADDITION OF WBC
FIG. 2. Effect ofpretreatment of E. coli 19+ on CL. E. coli organisms were pretreated with formaldehyde and heated as described in the text. The bacteria-toWBC ratio was 300:1. Each curve represents mean CL values from duplicate test vials during one of two similar experiments.
FIG. 3. CL from WBCs incubated with different cultures of E. coli 19. The bacteria-to- WBC ratio was 300:1. The PBS control lacked bacteria. Each curve represents mean CL values from duplicate test vials during a single experiment. E. coli 19 organisms were serially subcultured as follows: four times in TSB at 37°C (0); three times in TSB at 370C, followed by a single subculture in TSB at 370C (LI); or four times in TSB at 370C (0). Control vials received PBS alone (). Each curve represents mean CL values from duplicate test vials during one of two similar experiments.
MS STIMULATION OF WBC CL BY E. COLI 1017
VOL. 26, 1979
7O60 150 10 50
O
10
.
20
30
.
40
50
6
30.1 w W~~~~~ 2
ulate CL even when tested in concentrations as high as 200 Ag/ml. Pretreatment of WBCs with purified pill. An attempts was made to block 19+-induced CL by pretreating the WBCs (5 x 106/ml) with purified 19+ pili (187.5 [Lg/ml) for 15 min at room temperature (R0). When stimulated by 19+, pili-pretreated WBCs yielded a slightly higher peak CL (40,250 counts per 20 s) as compared with PBS-pretreated WBCs (38,000 counts per 20 s). Similar results were obtained 35
W
z 225
~~~~~~~~300-1 with 10mnM aMM
10
25X m DEXTROSE
a20
CONTROL Z
0
10
40 50 30 MIN AFTER ADDITION OF WBC
20
60
FIG. 4. CL from WBCs incubated with increasing concentrations of E. coli 19+. Bacteria-to- WBC ratios are shown for each curve. The unstimulated (PBS) control lacked E. coli. Each curve represents CL values from a single test vial during one of three representative experiments.
As little as 10 mM aMM completely inhibited CL stimulation by 19+ (Fig. 4). Neither dextrose nor sucrose (50 mM) blocked CL, whereas an equal concentration of aMM prevented CL (Fig. 5). In fact, 25 mM dextrose stimulated the CL burst elicited by 19+. The effect of aMM on CL stimulated by opsonized zymosan particles was also tested. Zymosan (5 mg/ml) caused peak CL of 211,500 counts per 20 s in the presence of 63.6 mM aMM and 224,500 counts per 20 s in the absence of aMM. WBCs incubated with aMM alone gave no CL. Thus, aMM does not appear to nonspecifically interfere with or stimulate WBC CL. Addition of aMM after E. coli association with WBCs. To determine whether CL can be stopped once the bacteria have associated with the WBCs, we added aMM to the test vials at various time intervals during the incubation. As shown in Fig. 6, the addition of 50 mM aMM within 5 min after the bacteria and WBCs were mixed completely prevented CL. After 5 min, the addition of aMM inhibited further increases in CL and caused a rapid decline in residual light emission. Purified pili. Several CL assays were done to determine whether purified 19+ and Bam+ pili could stimulate CL. However, pili failed to stim-
mM DEXTROSE
'j 5
5O/m
mm a MM 25mM aRM
50
10
D
o
SUCROSE
25 nM SUCROSE
UT
5. 5-
10
20
50 40 30 MIN AFTER ADDITION OF WBC
60
FIG. 5. Effect of saccharides on CL. E. coli Bam+ organisms were killed with 1% formaldehyde before the assay. The bacteria-to-WBC ratio was 180:1. Each curve represents the CL values from a single test vial during one of three similar experiments. 0
0
-t
I?z z -i
IL 0
10
20
50 60 40 30 MIN AFTER ADDITION OF WBC
70
FIG. 6. Addition of aMM at intervals during the incubation of WBCs with E. coli Bam+. E. coli organisms were killed with 1% formaldehyde before the assay. The bacteria-to-WBC ratio was 180:1. At the times indicated above each curve, aMM in PBS (0.27 ml) was added to the vials to a final concentration of 50 mM. The control suspension contained an equal volume of PBS added at zero time. Each curve represents the CL values from a single test vial during one of two representative experiments.
1018 MANGAN AND SNYDER with WBCs pretreated with purified Bam+ pili (133.3 ,ug/ml) and stimulated with Bam+. Pilipretreated and control WBCs failed to emit CL when incubated with 19- or Bam-. Unstimulated WBCs pretreated with pili did not emit CL (see above). DISCUSSION In previous studies, piliated E. coli with MS adhesins associated with and were killed by human WBCs in the presence of serum opsonins (22, 25). We further showed that piliated, but not nonpiliated, E. coli organisms caused a release of lysosomal enzymes from the WBCs without damaging the WBCs (22). In this communication, we extend our observations on the E. coli-WBC interaction by reporting that the association of these bacteria with the WBCs activates oxidative metabolism in the WBCs as detected by enhanced CL. E. coli organisms which lack MS adhesins failed to stimulate CL. WBCs incubated with E. coli 19- and challenged with strain 19+ yielded a CL burst of intensity equal to that obtained from WBCs incubated with PBS and stimulated with 19+. This indicates that the absence of CL from WBCs incubated with E. coli organisms without MS adhesins resulted from a lack of stimulation rather than from an inhibition or quenching of CL. WBCs incubated with 42+ yielded CL and could be further stimulated by 19+ to yield additional CL. This suggests that 19+ cultures either have more CL-stimulating cells or have cells with quantitatively more CL-stimulating activity than do 42+ cultures. This result also indicates that WBCs can detect a quantitative difference in CL-stimulating activity in E. coli cultures and respond to further stimulation with a second burst of CL. Concanvalin A (ConA), a plant lectin, binds to MS receptors on WBCs (21) and stimulates oxygen metabolism (24) and CL (unpublished data). Thus, one might speculate that the stimulation of WBC respiratory activity by ConA and E. coli MS adherence factors is mediated through common receptors by similar mechanisms.
Many of the biological effects associated with ConA appear to result from the ability of this multivalent lectin to aggregate mobile glycoprotein receptors into micropatches on the surface of the cells (11, 14). Clustering of ConA receptors on phagocytes has been noted (5), and Romeo et al. (24) speculated that the formation of receptor patches activates the oxidase enzyme involved in initiating the respiratory burst. In a similar fashion, E. coli organisms with MS adhesins may bind to several receptors simultane-
INFECT. IMMUN.
ously in one small area of the WBC surface and form a receptor patch. Isolated type 1 pili were unable to stimulate CL. The orientation of the pili during adherence to the WBC surface is unknown, but may be in such a manner that they fail to form the necessary clustering of receptors needed to trigger the oxidase enzyme. Alternatively, E. coli organisms may possess a second MS adhesin which mediates the association with WBCs and activates CL. Our prior finding that type 1 pili failed to inhibit the attachment of E. coli to WBCs (22), coupled with the date in this paper which show that CL stimulation by piliated E. coli is not inhibited to pili, tends to support the conclusion of a second adhesin. In this regard, Silverblatt et al. (25) obtained a poor correlation between the number of pili on several E. coli strains and the susceptibility of these bacteria to phagocytosis. Furthermore, Eshdat et al. (16) have recently isolated a mannose-specific lectin from E. coli which is functionally identical to type 1 pili, but which has an amino acid composition and subunit molecular weight which are different from type 1 pili. Whether our strains of E. coli possess this bacterial lectin and the role that the lectin has in the association of E. coli with WBCs remain to be determined. D-Mannose and aMM reversibly inhibit MS adherence of E. coli to eucaryotic cells (23). We found that aMM added to Bam+-WBC suspensions incubated at 370C for up to 20 min immediately interrupted CL, and subsequent light emission rapidly returned to control levels. Romeo and associates (24) reported that the oxidative stimulation of phagocytes by ConA could likewise to immediately reversed by the addition of a-methyl-D-glucopyranoside, a saccharide which reversibly inhibits ConA adherence. Therefore, these reports, suggest that the continual attachment of the stimulus to the WBC surface is required for the activation of the respiratory burst. In previous studies (22), we found that after incubation at 370C for 15 min, many E. coli organisms which associate with WBCs are phagocytized (ingested) and are not susceptible to displacement by the aMM treatment. The data from the CL and ingestion studies, therefore, suggest that only E. coli attached to and pertubating the outer membrane of the WBC participates in the stimulation of CL. This conclusion supports the evidence which indicates that CL results from the stimulation of a surface enzyme (12, 17, 18). Furthermore, the data suggest that the E. coli organisms internalized before the addition of aMM are incapable of triggering the oxidase enzyme responsible for initiating the respiratory burst. An explanation for this is presently unavailable, but it may be
MS STIMULATION OF WBC CL BY E. COLI 1019
VOL. 26, 1979
that the oxidase enzyme either may not be internalized as the cell membrane invaginates during phagocytosis or may be inactive when internalized. Further studies are necessary to determine the mechanism by which the oxidase enzyme is activated during the adherence of E. coli to MS receptors on the WBC membrane. Additional characterization of the MS adhesins on E. coli will undoubtedly lead to a better understanding of the E. coli-WBC interaction. More importantly, this particular bacteriumphagocyte relationship deserves special attention because (i) it occurs in the absence of serum opsonins, (ii) it involves a specific interaction between a bacterial adherence factor and a eucaryotic cell receptor, and (iii) it is readily reversible by a low concentration of aMM. The E. coli-WBC interaction system may, therefore, be a very useful tool for studying the cellular events involved in phagocytosis and the stimulation of CL. ACKNOWLEDGMENTS This work was supported by Biomedical Research Support Grant SO 7 RR 05433 17 and by the West Virginia University Medical Corp. The secretarial assistance of Elizabeth Rocovich is gratefully acknowledged.
LITERATURE CITED 1. Allen, R. C., R. L. Stjernholm, and R. H. Steele. 1972. Evidence for the generation of an electronic excitation state(s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem. Biophys. Res. Commun. 47:679-684. 2. Allen, R. C., S. J. Yevich, R. W. Orth, and R. H. Steele. 1974. The superoxide anion and singlet molecular oxygen: their role in the microbicidal activity of the polymorphonuclear leukocyte. Biochem. Biophys. Res. Commun. 60:909-917. 3. Babior, B. M., R. S. Kipnes, and J. T. Curnutte. 1973. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J. Clin. Invest. 52:741-744. 4. Bar-Shavit, Z., I. Ofek, R. Goldman, D. Mirelman, and N. Sharon. 1977. Mannose residues on phagocytes as receptors for the attachment of Escherichia coli and Salmonella typhi. Biochem. Biophys. Res. Commun. 78:455-460. 5. Berlin, R. 1972. Effect of Concanavalin A on phagocytosis. Nature (London) New Biol. 235:44-45. 6. Boyum, A. 1968. Isolation of leukocytes from human blood: further observations. Methylcellulose, dextran, and ficoll as erythrocyte-aggregating agents. Scand. J. Clin. Lab. Invest. 21(Suppl. 9):31-50. 7. Brinton, C. C., Jr. 1965. The structure, function, synthesis, and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram negative bacteria. Trans. N.Y. Acad. Sci. 27:1003-1054. 8. Brinton, C. C., Jr. 1967. Contributions of pili to the specificity of the bacterial surface, and a unitary hy-
pothesis of conjugal infectious heredity, p. 37-70. In B. D. Davis and L. Warren (ed.), The specificity of cell surfaces. Prentice-Hall, Inc., Englewood Cliffs, N.J. 9. Cheson, B. D., R. J. Christensen, R. Sperling, B. E. Kohler, and B. M. Babior. 1976. The origin of the chemiluminescence of phagocytosing granulocytes. J. Clin. Invest. 58:789-796. 10. Clark, R. A., and H. R. Kimball. 1971. Defective granulocyte chemotaxis in the Chediak-Higashi syndrome. J. Clin. Invest. 50:2645-2652. 11. Cunningham, B. A., B. A. Sela, I. Yahara, and G. M. Edelman. 1976. Structure and activities of lymphocyte mitogens, p. 13-30. In J. J. Openheim and D. L. Rosenstreich (ed.), Mitogens in immunology. Academic Press Inc., New York. 12. Dewald, B., M. Gaggiolini, J. T. Curnutte, and B. M. Babior. 1979. Subcellular localization of the superoxide-forming enzyme in human neutrophils. J. Clin. Invest. 63:21-29. 13. Duguid, J. P., and R. R. Gillies. 1957. Fimbriae and adhesive properties in dysentery bacilli. J. Pathol. Bacteriol. 74:397-411. 14. Edelman, G. M., B. A. Cunningham, G. N. Reeke, Jr., J. W. Becker, M. J. Waxdal, and J. L. Want. 1972. The covalent and three-dimensional structure of Concanavalin A. Proc. Natl. Acad. Sci. U.S.A. 69:25802584. 15. Edwards, P. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed., p. 21-47. Burgess Publishing Co., Minneapolis, Minn. 16. Eshdat, Y., I. Ofek, Y. Yashour-Gan, N. Sharon, and D. Mirelman. 1978. Isolation of a mannose-specific lectin from Escherichia coli and its role in the adherence of the bacteria to epithelial cells. Biochem. Biophys. Res. Commun. 85:1551-1559. 17. Goldstein, I. M., M. Cerqueria, S. Lind, and H. B. Kaplan. 1977. Evidence that the superoxide-generating system of human leukocytes is associated with the cell surface. J. Clin. Invest. 59:249-254. 18. Goldstein, L. M., D. Roos, H. B. Kaplan, and G. Weissmann. 1975. Complement and immunoglobulins stimulate superoxide production by human leukocytes independently of phagocytosis. J. Clin. Invest. 56:11551163. 19. Karnovsky, M. L. 1962. Metabolic basis of phagocytic activity. Physiol. Rev. 42:143-168. 20. Klebanoff, S. J. 1975. Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Semin. Hematol. 12:117-142. 21. iUs, H., and N. Sharon. 1973. The biochemistry of plant lectins (phytohemagglutinins). Annu. Rev. Biochem. 42:541-574. 22. Mangan, D. F., and L. S. Snyder. 1979. Mannose-sensitive interaction of Escherichia coli with human peripheral leukocytes in vitro. Infect. Immun. 26:520-527. 23. Ofek, I., E. H. Beachey, and N. Sharon. 1978. Surface sugars of animal cells as determinants of recognition in bacterial adherence. Trends Biochem. Sci. 3:159-160. 24. Romeo, D., G. Zabucchi, and F. Rossi. 1973. Reversible metabolic stimulation of polymorphonuclear leukocytes and macrophages by Concanavalin A. Nature (London) New Biol. 243:111-112. 25. Silverblatt, F. J., J. S. Dreyer, and S. Schauer. 1979. Effect of pili on susceptibility of Escherichia coli to phagocytosis. Infect. Immun. 24:218-223. 26. Stossel, T. P. 1974. Phagocytosis. N. Engl. J. Med. 290: 717-724, 774-780, 833-838.
